Evaluation of copolymer diethylamide

ABSTRACT

Methods of analyzing glatiramer acetate (GA) or a polymeric precursor thereof are provided. The methods can include determining a level of one or more diethylamide-modified amino acids in a sample comprising GA or a polymeric precursor thereof, and selecting at least a portion of the sample based on the assessment of the one or more diethylamide-modified amino acids in the sample.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/692,490, filed Dec. 3, 2012, which is a continuation of U.S.application Ser. No. 13/584,309, filed Aug. 13, 2012, which claimspriority under 35 U.S.C. 120 to U.S. Provisional Application Ser. Nos.61/506,494, filed Jul. 11, 2011; 61/528,477, filed Aug. 29, 2011, and toInternational Patent Application no. PCT/US2012/046270, filed Jul. 11,2012, all of which are herein incorporated by reference in theirentireties.

TECHNICAL FIELD

The present disclosure relates to methods for making (e.g.,manufacturing or producing) glatiramer acetate (GA), including assessingthe distribution of diethylamide (DEA) in GA and/or polymeric precursorsof GA.

BACKGROUND

Glatiramer acetate (GA), marketed commercially as COPAXONE®, consists ofthe acetate salts of synthetic polypeptides containing four naturallyoccurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, andL-lysine with a reported average molar fraction of 0.141, 0.427, 0.095,and 0.338, respectively. Chemically, GA is designated L-glutamic acidpolymer with L-alanine, L-lysine and L-tyrosine, acetate (salt). Itsstructural formula is:

(Glu, Ala, Lys, Tyr)_(x).xCH₃COOH(C₅H₉NO₄.C₃H₇NO₂.C₆H₁₄N₂O₂.C₉H₁₁NO₃)_(x) .xC₂H₄O₂CAS—147245-92-9

Other than molecular weight and amino acid composition, which arespecified in the approved label for the product, the label and otheravailable literature for COPAXONE® does not provide detailed informationabout the physiochemical characteristics of the product.

SUMMARY

The present disclosure provides methods for using the distribution ofdiethylamide (DEA) in Glatiramer Acetate (GA) and/or polymericprecursors of GA and/or mother liquor resulting from the manufacture ofGA in the selection of GA (e.g., upon completion of a manufacturingprocess) and/or polymeric precursors of GA (e.g., during a manufacturingprocess); to determine or confirm compliance of GA and/or polymericprecursors of GA with industrial and/or regulatory standards; to assessor confirm manufacturing consistency; as a quality control standard foruse during manufacturing and/or against GA. In some embodiments, methodsinclude assessing (e.g., measuring, analyzing, detecting, determining,evaluating, estimating, predicting, monitoring, reviewing, and/orcorrelating) DEA distribution in Glatiramer Acetate (GA) and/orpolymeric precursors of GA and/or mother liquor resulting from themanufacture of GA. Additional applications will be apparent to those ofskill in the art based on the disclosure herein.

In some embodiments, the disclosure provides methods for selecting abatch of a composition comprising GA or a polymeric precursor thereof(e.g., referred to as selection methods). These selection methods caninclude: providing or obtaining a sample of a batch of a compositioncomprising GA or a polymeric precursor thereof; determining the relativelevel or ratio of diethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample; and selecting the batch if (e.g., and only if)the relative level or ratio of diethylamide-modifiedalanine:diethylamide-modified lysine:diethylamide-modified glutamicacid:diethylamide-modified tyrosine in the sample is about59.5-76.1%:11.3-17.3%:9.9-15.0%:4.8-7.2%, wherein the total of therelative levels or ratio is 100%. In other embodiments the batch isselected if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 45-95%:9-22%:7-19%:4-9%, wherein thetotal of the relative levels or ratio is 100%. In other embodiments thebatch is selected if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 54-84%:10-19%:9-17%:4-8%, wherein thetotal of the relative levels or ratio is 100%. In other embodiments thebatch is selected if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 60-76%:11-17%:10-15%:5-7%, wherein thetotal of the relative levels or ratio is 100%.

The present disclosure also provides identification methods that can beused to identify a copolymer as glatiramer acetate. In some cases thecopolymer subjected to the identification methods is a copolymer ofglutamic acid, alanine, tyrosine, and lysine with a reported averagemolar fraction of 0.141, 0.427, 0.095, and 0.338, respectively andhaving a peak average molecular weight of 5,000-7,000. These selectionmethods can include: providing or obtaining a sample of a batch of acomposition comprising a copolymer (e.g., a copolymer of alanine,lysine, glutamic acid and tyrosine); determining the relative level orratio of diethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample; and identifying the copolymer as glatirameracetate if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about59.5-76.1%:11.3-17.3%:9.9-15.0%:4.8-7.2%, wherein the total of therelative levels or ratio is 100%. In other embodiments the copolymer isidentified as glatiramer acetate if (e.g., and only if) the relativelevel or ratio of diethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 45-95%:9-22%:7-19%:4-9%, wherein thetotal of the relative levels or ratio is 100%. In other embodiments thecopolymer is identified as glatiramer acetate if (e.g., and only if) therelative level or ratio of diethylamide-modifiedalanine:diethylamide-modified lysine:diethylamide-modified glutamicacid:diethylamide-modified tyrosine in the sample is about54-84%:10-19%:9-17%:4-8%, wherein the total of the relative levels orratio is 100%. In other embodiments the copolymer is identified asglatiramer acetate if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 60-76%:11-17%:10-15%:5-7%, wherein thetotal of the relative levels or ratio is 100%.

In other embodiments, selection methods can include: providing a sampleof a batch of a composition comprising GA or a polymeric precursorthereof; determining (e.g., measuring) a level of one or more ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample; and selecting the batch if (e.g., and only if) the levelof the one or more of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample conforms to a predeterminedreference value. In some aspects, the batch is selected if (e.g., andonly if): the level of two or more of diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample is about equal to a level shown in Table 1: the level ofthree or more of diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine in the sample is aboutequal to a level or ratio thereof shown in Table 1; and/or the level ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor shown in Table 1.

In other embodiments, the identification methods can include: providinga sample of a batch of a composition comprising a copolymer (e.g., acopolymer of alanine, lysine, glutamic acid and tyrosine); determining(e.g., measuring) a level of one or more of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample; and identifyingthe copolymer as glatiramer acetate if (e.g., and only if) the level ofthe one or more of diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and diethylamide-modifiedtyrosine in the sample conforms to a predetermined reference value. Insome aspects, the copolymer is identified as glatiramer acetate (e.g.,and only if): the level of two or more of diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample is about equal to a level shown in Table 1: the level ofthree or more of diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine in the sample is aboutequal to a level or ratio thereof shown in Table 1; and/or the level ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor shown in Table 1.

In further embodiments, selection methods can include: providing asample of a batch of a composition comprising GA or a polymericprecursor thereof; determining a level of diethylamide-modified alaninein the sample; and selecting the batch if (e.g., and only if): the levelof diethylamide-modified alanine in the sample is at least about 45% ofthe total diethylamide-modified amino acids in the sample; the level ofdiethylamide-modified alanine in the sample is at least about 50% of thetotal diethylamide-modified amino acids in the sample; and/or the levelof diethylamide-modified alanine in the sample is at least about 65% ofthe total diethylamide-modified amino acids in the sample. In someaspects, these methods can further include determining a level of one ormore of diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample; and selectingthe batch if (e.g., and only if): the level of the one or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelshown in Table 1 or if the ratio of two or more of diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and diethylamide-modifiedtyrosine is about equal to a ratio shown in Table 1; the level of two ormore of diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample is about equal toa level shown in Table 1; and/or the level of three or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor ratio thereof shown in Table 1.

In some embodiments, selection methods can include: providing a sampleof a batch of a composition comprising GA or a polymeric precursorthereof; determining the relative level or ratio of two or more ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample; and selecting the batch if (e.g., and only if): therelative level or ratio of the two or more of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample conforms to apredetermined reference value; the relative level or ratio of three ormore of diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample conforms to a predetermined reference value; the relativelevel or ratio of diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and diethylamide-modifiedtyrosine in the sample conforms to a predetermined reference value. Insuch embodiments, the or a predetermined reference value can be: arelative level of diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and/ordiethylamide-modified tyrosine shown in Table 1; a relative level orratio of diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosineof about 10:2:2:1.

For the selection and identification methods disclosed herein,determination or determining can include cleaving the GA or thepolymeric precursor thereof in the sample, e.g., to yield a samplecomprising fragments of the GA or fragments of the precursor. Fragmentsresulting from such cleavage can include diethylamide-modified alanine(Ala-DEA) diethylamide-modified lysine (Lys-DEA), diethylamide-modifiedglutamic acid (Glu-DEA), and/or diethylamide-modified tyrosine(Tyr-DEA). Such fragments can be or can include 1-mers (e.g., can be asingle amino acid with a C-terminal DEA group or can be peptidescontaining a DEA-modified C-terminal amino acid). For example, fragmentscan include: a second amino acid selected from alanine, lysine, glutamicacid, and tyrosine; three amino acids, wherein the second and thirdamino acids are independently selected from alanine, lysine, glutamicacid, and tyrosine; and/or up to 10 amino acids, wherein the amino acidsare independently selected from alanine, lysine, glutamic acid, andtyrosine. In any case, a DEA modified peptide will be identified by itsC-Terminal-DEA modified amino acid. For example, peptide‘Lys-Lys-Ala-DEA’ will be identified as Ala-DEA. In some aspects,selection methods can include a step of isolating or removingdiethylamine such that the sample comprising fragments of the GA orfragments of the precursor is substantially free of diethylamine.

Where selection methods include a determining step requiring cleavage ofGA or polymeric precursors thereof, such cleavage can be performedenzymatically, chemically, or using physical methods. Exemplary cleavagemethods can include contacting the sample with one, two, three, or moreproteases (e.g., selected from trypsin, chymotrypsin, elastase, ficin,papain, pepsin, plasmin, thermolysin, endopeptidase, proteinase K, oxbile, lemon pectin, horseradish peroxidase, glu-c, endo lys-C,carboxypeptidase, calpain, and subtilisin) under conditions and for atime sufficient to yield fragments of the GA or fragments of theprecursor that include diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine. In some aspects, at least one proteaseis proteinase K.

For the selection methods, determination or determining can furtherinclude adding to the sample known concentrations of detectable aminoacid or peptide standards that include diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine. These detectable amino acid or peptidestandards be DEA-modified and can include one, two, three, more thanthree amino acids, or any number of amino acids required to representthe fragments generated or expected to be generated from the cleavage.In some aspects, detectable amino acids or peptide standards can beisotopically labeled.

Where selection methods include cleavage and addition or use of knownconcentrations of detectable amino acid or peptide standards, detectioncan include: detecting peptides corresponding to the detectable aminoacid or peptide standards and the detectable amino acid or peptidestandards to determine the total amount of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine containing amino acids peptidesin the sample; subtracting the known concentrations of the detectableamino acid or peptide standards to determine the levels ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample; and, optionally, determining the sum ofdiethylamide-modified alanine amino acids or peptide, the sum ofdiethylamide-modified lysine amino acids or peptide, the sum ofdiethylamide-modified glutamic acid amino acids or peptide, and the sumof diethylamide-modified tyrosine containing amino acids and peptides.Detecting peptides, e.g., as part of the detection step, can include:separating diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining amino acids and peptides and the detectable amino acid orpeptide standards using chromatography to yield chromatographicallyseparated samples comprising diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine containing amino acids and peptides andthe detectable amino acid or peptide standards; and detecting thechromatographically separated samples. Chromatographically separatedsamples can be assessed, measured, or evaluated using mass spectroscopyanalysis. Such mass spectroscopy can include MRM detection or tandemmass spectrophotometry (MS/MS) analysis. Alternatively or in addition,chromatographically separated samples can be assessed, measured, orevaluated using nuclear magnetic resonance (NMR) analysis, infraredspectroscopy, gel electrophoresis, emission spectroscopy, UV-visspectroscopy, Raman spectroscopy, and antibody detection.

For the selection methods, selection of a batch if (e.g., and only if)the various qualifying parameters are met can include: using at least aportion of the batch in the manufacture or preparation of a GA drugproduct; formulating at least a portion of the batch for administrationto a subject; formulating at least a portion of the batch for injection;and/or releasing at least a portion of the batch for pharmaceutical use.If a batch does not satisfy the various qualifying parameters, then thebatch can be withheld and/or discarded.

In some embodiments, selection methods can include: providing a sampleof a batch comprising GA or a polymeric precursor thereof; contactingthe sample with proteinase K for a time and under conditions sufficientto yield a second sample comprising fragments of the GA or fragments ofthe precursor, wherein the fragments include diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine; adding to the second sampleknown concentrations of detectable amino acid or peptide standards thatrepresent diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, or diethylamide-modified tyrosinecontaining amino acids or peptides; chromatographically separatingdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining amino acid or peptides and the detectable amino acid orpeptide standards to yield chromatographically separated samplescomprising diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining amino acids and peptides and the detectable amino acid orpeptide standards; detecting the chromatographically separated samples;subtracting the known concentrations of the detectable standards todetermine the levels of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine; determining the sum ofdiethylamide-modified alanine amino acids or peptide, the sum ofdiethylamide-modified lysine amino acids or peptide, the sum ofdiethylamide-modified glutamic acid amino acids or peptide, and the sumof diethylamide-modified tyrosine amino acids or peptide; and selectingthe batch and optionally using at least a portion of the batch in amanufacturing process for GA if the ratio of the sums of two or more,three or more, or all four of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample are about equal to theratio between two or more, three or more, or all four ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosineshown in Table 1.

In some embodiments, the disclosure provides methods for analyzing abatch of a composition comprising GA or a polymeric precursor thereof(e.g., referred to as analysis methods). These analysis methods caninclude detection and selection steps or methods disclosed above for theselection methods. In some aspects, selection methods can include:detecting a level of diethylamide-modified alanine in a sample of abatch of a composition comprising GA or a polymeric precursor thereof;and determining if the level of diethylamide-modified alanine in thesample is at least about 45%, 50%, or 55% of the totaldiethylamide-modified amino acids in the sample. Alternatively or inaddition, analysis methods can include: detecting a level of one or moreof diethylamide-modified lysine, diethylamide-modified glutamic acid,and diethylamide-modified tyrosine in the sample; and determining if thelevel of the diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine in the sample is aboutequal to a level shown in Table 1 or if the ratio of two or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine is about equal to a ratio shown in Table1.

In some embodiments, analysis methods can include: detecting a level ofone or more of diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and diethylamide-modifiedtyrosine in a sample of a batch of a composition comprising GA or apolymeric precursor thereof, and determining if the level of: one ormore of diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample is about equal toa level shown in Table 1; two or more of diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample is about equal to a level or ratio shown in Table 1; threeor more of diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample is about equal toa level or ratio shown in Table 1; and/or diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample is about equal to a level or ratio shown in Table 1.

In other embodiments, analysis methods can include: detecting therelative level or ratio of diethylamide-modifiedalanine:diethylamide-modified lysine:diethylamide-modified glutamicacid:diethylamide-modified tyrosine in a sample of a batch of acomposition comprising GA or a polymeric precursor thereof; anddetermining if the relative level or ratio of diethylamide-modifiedalanine:diethylamide-modified lysine:diethylamide-modified glutamicacid:diethylamide-modified tyrosine in the sample is about45-95%:9-22%:7-19%:4-9% wherein the total of the relative levels orratio is 100%.

In further embodiments, analysis methods can include: detecting therelative level or ratio of two or more of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in a sample of a batch of a compositioncomprising GA or a polymeric precursor thereof, and determining if therelative level or ratio of: two or more of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample conforms to apredetermined reference value; three or more of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample conforms to apredetermined reference value; and/or diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample conforms to a predeterminedreference value. In such methods, the predetermined reference value is arelative level of diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and/ordiethylamide-modified tyrosine shown in Table 1. In some instances, thepredetermined reference value can be a relative level or ratio ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosineof about 10:2:2:1.

In some aspects, analysis methods can include cleaving the GA or thepolymeric precursor thereof in the sample to yield or generate a samplecomprising fragments of the GA or fragments of the precursor, whereinthe fragments include diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, ordiethylamide-modified tyrosine. Methods can further include removingdiethylamine such that the sample comprising fragments of the GA orfragments of the precursor is substantially free of diethylamine.

For the analysis methods, determination or determining can furtherinclude adding to the sample known concentrations of detectable aminoacid or peptide standards that include diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine. Such detectable standards can beisotopically labeled. In some aspects, detecting peptides correspondingto the detectable amino acid or peptide standards and the detectableamino acid or peptide standards to determine the total amount ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining amino acids or peptides in the sample; and subtracting theknown concentrations of the detectable amino acid or peptide standardsto determine the levels of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample. Methods can furtherinclude determining the sum of diethylamide-modified alanine amino acidsor peptide, the sum of diethylamide-modified lysine amino acids orpeptide, the sum of diethylamide-modified glutamic acid amino acids orpeptide, and the sum of diethylamide-modified tyrosine amino acids orpeptide. Where known concentrations of detectable amino acid or peptidestandards are used, detection can include: separatingdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining amino acids and peptides and the detectable amino acid orpeptide standards using chromatography to yield chromatographicallyseparated samples comprising diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine containing peptides and the detectableamino acid or peptide standards; and detecting the chromatographicallyseparated samples.

In some embodiments, the disclosure provides methods of manufacturing GAdrug product (e.g., referred to as manufacturing methods). Thesemanufacture methods can include detection and selection steps or methodsdisclosed above for the selection and/or analysis methods. In someaspects, such methods can include: polymerizing N-carboxy anhydrides ofL-alanine, benzyl-protected L-glutamic acid, trifluoroacetic acid(TFA)-protected L-lysine, and L-tyrosine to generate a protectedcopolymer; treating the protected copolymer to partially depolymerizethe protected copolymer and deprotect benzyl protected groups;deprotecting TFA-protected lysines to generate GA; purifying the GA;detecting a level of diethylamide-modified alanine in a sample of thepurified GA; and using the GA in the manufacture or preparation of a GAdrug product if (e.g., if and only if) the level ofdiethylamide-modified alanine in the sample is at least about 45%, 55%,or 65% of the total diethylamide-modified amino acids in the sample. Insome aspects, there methods can further include: detecting a level ofone or more of diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine in the sample; andusing the GA in the manufacture or preparation of a GA drug product if:the level of the one or more of diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample is about equal to a level shown in Table 1 or if the ratioof two or more of diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine is about equal to aratio shown in Table 1; the level of the two or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelshown in Table 1; and/or the level of the three or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor ratio thereof shown in Table 1.

In some embodiments, manufacturing methods can include: polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a protected copolymer; treating the protected copolymer topartially depolymerize the protected copolymer and deprotect benzylprotected groups; deprotecting TFA-protected lysines to generate GA;purifying the GA; detecting a level of one or more ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein a sample of the purified GA; and using the GA in the manufacture orpreparation of a GA drug product if (e.g., if and only if) the level ofone or more of diethylamide-modified lysine, diethylamide-modifiedglutamic acid, and diethylamide-modified tyrosine in the sample is aboutequal to a level shown in Table 1, and/or if the ratio of two or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine is about equal to a ratio shown inTable 1. In some aspects, these methods can include: using the GA in themanufacture or preparation of a GA drug product if the level of two ormore of diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine in the sample is about equal toa level shown in Table 1; using the GA in the manufacture or preparationof a GA drug product if the level of three or more ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor ratio thereof shown in Table 1; and/or using the GA in themanufacture or preparation of a GA drug product if the level ofdiethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample is about equal to a levelor ratio thereof shown in Table 1.

In other embodiments, manufacturing methods can include: polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a protected copolymer; treating the protected copolymer topartially depolymerize the protected copolymer and deprotect benzylprotected groups; deprotecting TFA-protected lysines to generate GA;purifying the GA; detecting the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in a sample of the purified GA; and using the GA in themanufacture or preparation of a GA drug product if the relative level orratio of diethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about59.5-76.1%:11.3-17.3%:9.9-15.0%:4.8-7.2%, wherein the total of therelative levels or ratio is 100%. In other embodiments the batch is usedif (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 45-95%:9-22%:7-19%:4-9%, wherein thetotal of the relative levels or ratio is 100%. In other embodiments thebatch is used if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 54-84%:10-19%:9-17%:4-8%, wherein thetotal of the relative levels or ratio is 100%. In other embodiments thebatch is used if (e.g., and only if) the relative level or ratio ofdiethylamide-modified alanine:diethylamide-modifiedlysine:diethylamide-modified glutamic acid:diethylamide-modifiedtyrosine in the sample is about 60-76%:11-17%:10-15%:5-7%, wherein thetotal of the relative levels or ratio is 100%.

In further embodiments, manufacturing methods can include: polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a protected copolymer; treating the protected copolymer topartially depolymerize the protected copolymer and deprotect benzylprotected groups; deprotecting TFA-protected lysines to generate GA;purifying the GA; detecting the relative level or ratio of two or moreof diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample; and using the GA in the manufacture or preparation of aGA drug product if the relative level or ratio of the two or more ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample conforms to a predetermined reference value. In someaspects, these methods can include: using the GA in the manufacture orpreparation of a GA drug product if the relative level or ratio of threeor more of diethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample conforms to a predetermined reference value; and/or usingthe GA in the manufacture or preparation of a GA drug product if therelative level or ratio of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine in the sample conforms to a predeterminedreference value. For these methods, the predetermined reference valuecan be a relative level of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid,and/or diethylamide-modified tyrosine shown in Table 1; and/or arelative level or ratio of diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine of about 10:2:2:1.

In some aspects, for the manufacturing methods, detecting DEA-modifiedamino acids can include cleaving the GA or the polymeric precursorthereof in the sample to yield a sample comprising fragments of the GAor fragments of the precursor, wherein the fragments includediethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosine.Detecting can also optionally include removing diethylamine, such thatthe sample comprising fragments of the GA or fragments of the precursoris substantially free of diethylamine. In some aspects, wherein GA orthe polymeric precursor are cleaved, cleavage can be performedenzymatically, chemically, and/or using physical methods. Whereenzymatic cleavage is used, methods can include contacting the samplewith one, two, or more proteases (e.g., one or more of trypsin,chymotrypsin, elastase, ficin, papain, pepsin, plasmin, thermolysin,endopeptidase, proteinase K, ox bile, lemon pectin, horseradishperoxidase, glu-c, endo lys-C, carboxypeptidase, calpain, and/orsubtilisin) under conditions and for a time sufficient to yieldfragments of the GA or fragments of the precursor that includediethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosine.In some instances, the one or more proteases can include proteinase K.Detecting can also include adding to the sample known concentrations ofdetectable amino acid or peptide standards (e.g., isotopically labeleddetectable amino acid or peptide standards) that includediethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosine.Where detectable amino acid or peptide standards are added, detectingpeptides can include detecting peptides corresponding to the detectableamino acid or peptide standards and the detectable amino acid or peptidestandards to determine the total amount of diethylamide-modifiedalanine, diethylamide-modified lysine, diethylamide-modified glutamicacid, and diethylamide-modified tyrosine containing amino acids orpeptides in the sample; and subtracting the known concentrations of thedetectable amino acid or peptide standards to determine the levels ofdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinein the sample. These methods can further optionally include determiningthe sum of diethylamide-modified alanine amino acids or peptide, the sumof diethylamide-modified lysine amino acids or peptide, the sum ofdiethylamide-modified glutamic acid amino acids or peptide, and the sumof diethylamide-modified tyrosine amino acids or peptide. Also, wheredetectable amino acid or peptide standards are used, detecting peptidescorresponding to the detectable amino acid or peptide standards and thedetectable amino acid or peptide standards can include: separatingdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining peptides and the detectable standards using chromatography toyield chromatographically separated samples comprisingdiethylamide-modified alanine, diethylamide-modified lysine,diethylamide-modified glutamic acid, and diethylamide-modified tyrosinecontaining peptides and the detectable standards; and detecting thechromatographically separated samples. In some aspects, massspectroscopy analysis can be used. For example, mass spectroscopy caninclude using MRM detection or tandem mass spectrophotometry (MS/MS).Chromatographically separated samples can also be detected, for exampleusing nuclear magnetic resonance (NMR) analysis, infrared spectroscopy,gel electrophoresis, emission spectroscopy, UV-vis spectroscopy, Ramanspectroscopy, and antibody detection.

In some instances, methods include of comparing the determined level toa GA reference standard. In some instances, the reference standard canbe or can include a commercially available pharmaceutical preparation ofGA (Copaxone®). In another embodiment, the reference standard is aspecification for commercial release of a drug product comprising GA.For example, the specification for commercial release can be thespecification provided by the U.S. Food & Drug Administration (FDA),e.g., for the pharmaceutical release of GA. In some instances, where thelevel of the at least one GA-induced polypeptide is within apredetermined range or has a preselected relationship with the referencevalue, the method can include: providing and/or receiving informationregarding the predetermined range or preselected relationship to anotherparty (e.g., a party manufacturing GA), classifying, selecting,accepting, discarding, releasing, or withholding a batch of GA;reprocessing a batch through a previous manufacturing step; processing abatch of GA into drug product, shipping the product from a batch of GA,moving the batch of GA to a new location; or formulating, labeling,packaging, selling, offering for sell, releasing a batch of GA intocommerce and/or directing any of the above actions.

In some embodiments, methods include of comparing the determined levelto a GA reference standard (e.g., a GA product description in an FDAlabel, a Physician's Insert, a USP monograph, or an EP monograph) toassess suitability for undergoing a next step, e.g., as disclosedherein. In some embodiments, the methods include recording thedetermined level in a print or computer-readable medium, e.g., in a testreport, Material Safety Data Sheet (MSDS) or Certificate of Testing orCertificate of Analysis (CofA).

In some embodiments, methods of manufacturing a drug product comprisingglatiramer acetate include obtaining a sample of a batch of glatirameracetate; measuring, in the sample of the batch, the level of at leastone individual diethylamide-modified amino acid selected from the groupconsisting of: diethylamide-modified alanine, diethylamide-modifiedlysine, diethylamide-modified glutamic acid, and diethylamide-modifiedtyrosine; and processing at least a portion of the batch of glatirameracetate to produce a drug product comprising glatiramer acetate if atleast one of the following measured criteria are met: (i) the level ofdiethylamide-modified alanine in the sample is 59.5-76.1% of the totaldiethylamide-modified amino acids in the sample on a mol percent basis;(ii) the level of diethylamide-modified lysine detected in the sample is11.3-17.3% of the total diethylamide-modified amino acids in the sampleon a mol percent basis; (iii) the level of diethylamide-modifiedglutamic acid detected in the sample is 9.9-15.0% of the totaldiethylamide-modified amino acids in the sample on a mol percent basis;and (iv) the level of diethylamide-modified tyrosine detected in thesample is 4.8-7.2% of the total diethylamide-modified amino acids in thesample on a mol percent basis, thereby manufacturing a drug productcomprising glatiramer acetate. In some instances, at least a portion ofthe batch of glatiramer acetate is processed to produce a drug productcomprising glatiramer acetate if at least two, or at least three, or allfour of the criteria are met.

In some instances, the methods of manufacturing a drug productcomprising glatiramer acetate include processing at least a portion ofthe batch of glatiramer acetate to produce a drug product if the levelof diethylamide-modified alanine in the sample is 59.5-76.1% of thetotal diethylamide-modified amino acids in the sample on a mol percentbasis.

In other instances, the methods of manufacturing a drug productcomprising glatiramer acetate include processing at least a portion ofthe batch of glatiramer acetate to produce a drug product if the levelof diethylamide-modified lysine in the sample is 11.3-17.3% of the totaldiethylamide-modified amino acids in the sample on a mol percent basis.In certain instances, methods of manufacturing a drug product comprisingglatiramer acetate include processing at least a portion of the batch ofglatiramer acetate to produce a drug product if the level ofdiethylamide-modified glutamic acid in the sample is 9.9-15.0% of thetotal diethylamide-modified amino acids in the sample on a mol percentbasis. In other instances, the methods of manufacturing a drug productcomprising glatiramer acetate include processing at least a portion ofthe batch of glatiramer acetate to produce a drug product if the levelof diethylamide-modified tyrosine in the sample is 4.8-7.2% of the totaldiethylamide-modified amino acids in the sample on a mol percent basis.

In other instances, the methods of manufacturing a drug productcomprising glatiramer acetate include processing the glatiramer acetateto produce a drug product if the following criteria are met: the levelof diethylamide-modified alanine detected in the sample is 59.5-76.1% ofthe total diethylamide-modified amino acids in the sample on a molpercent basis; the level of diethylamide-modified lysine detected in thesample is 11.3-17.3% of the total diethylamide-modified amino acids inthe sample on a mol percent basis; the level of diethylamide-modifiedglutamic acid detected in the sample is 9.9-15.0% of the totaldiethylamide-modified amino acids in the sample on a mol percent basis;and the level of diethylamide-modified tyrosine detected in the sampleis 4.8-7.2% of the total diethylamide-modified amino acids in the sampleon a mol percent basis.

In some embodiments, the step of processing the batch of glatirameracetate includes combining at least a portion of the glatiramer acetatein the batch with a pharmaceutically acceptable carrier or excipient.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF FIGURES

FIG. 1 is a flow diagram providing an exemplary embodiment of theprocess disclosed herein. Hexagons represent pyroglutamate. Diamondsrepresent diethylamide. Circles represent distinct amino acids.

DETAILED DESCRIPTION

Processes for the manufacture of Glatiramer Acetate (GA) generallyinclude:

Polymerization of N-carboxy anhydrides of L-alanine, benzyl-protectedL-glutamic acid, trifluoroacetic acid (TFA) protected L-lysine andL-tyrosine (collectively referred to as NCAs) to result in a protectedcopolymer (Intermediate-1);

Depolymerization and benzyl deprotection of Intermediate-1 using, forexample, hydrobromic acid in acetic acid (e.g., phenol treated 33%HBr/acetic acid) to generate Intermediate-2; and

Deprotection of the TFA-protected lysines on Intermediate-2 (e.g., bytreatment with piperidine) to create Intermediate-3, followed byprocessing to generate GA and further purification and drying of theisolated GA drug substance.

During polymerization, NCAs are co-polymerized in a predetermined ratiousing diethylamine as an initiator. This addition of diethylamine to thereaction mixture results in modification of the C-terminus of a portionof amino acids in the reaction mixture to include diethylamide (DEA).Upon consumption of the NCA components, the reaction mixture is quenchedin water. The resulting protected polymer (Intermediate-1) is isolatedand dried. During depolymerization and benzyl deprotection,Intermediate-1 is treated with phenol-treated 33% HBr in acetic acid(HBr/AcOH). This results in the cleavage of the benzyl protecting groupon the glutamic acids as well as cleavage of peptide bonds throughoutthe polymer. After a period of time the reaction is quenched with water,and the product polymer is isolated by filtration and washed with water.The product polymer, Intermediate-2, has a reduced molecular weightrelative to Intermediate-1. Intermediate-2 is dried before proceeding todeprotection of TFA-protected lysine. During deprotection TFA-protectedlysines, Intermediate-2 is treated with aqueous piperidine to remove thetrifluoroacetyl group on the lysine. The resulting copolymer,Intermediate-3, is subsequently purified usingdiafiltration/ultrafiltration and the resulting acetate salt is dried toproduce Glatiramer Acetate drug substance. Exemplary methods for themanufacture of GA are known in the art (see, for example, U.S. Pat. No.3,849,550; WO 95/031990, US 2006/0154862, US 2007/0021324, US2010/0256039, US 2007/0021324, US 2009/0263347, and US 2010/0256039, andWO 2010/017292 which are hereby incorporated by reference in theirentirety).

As disclosed herein, there are certain detectable attributes of GA thatare conserved from batch-to-batch. These attributes can be used, e.g.,to select GA or polymeric precursors of GA and/or to monitor, assess,and/or evaluate GA process and/or batch quality.

Based on detailed characterization of GA and the GA production process,the present disclosure provides that diethylamide (DEA) distribution onthe C-terminus of a portion of amino acids of GA is a conserveddetectable attribute, a characteristic, hallmark, and/or a signature(e.g., a structural signature) of GA and/or the GA production process(e.g., polymeric precursors of GA). Accordingly, methods are describedherein for assessing or evaluating DEA distribution in GA and/orpolymeric precursors of GA, e.g., as a means for selecting GA and/orpolymeric precursors of GA.

In some embodiments, methods are described herein for observing DEAdistribution in GA and/or polymeric precursors of GA and taking actionwith respect to the GA and/or polymeric precursors on the basis of theobserved DEA distribution therein. For example, methods can includeobserving data related to DEA distribution in GA and/or polymericprecursors of GA (e.g., electronic data, stored data, and/or printeddata) as part of a process for the selection, review (e.g., qualitycontrol), and/or manufacture of GA and/or polymeric precursors of GA.Alternatively or in addition, methods can include assessment of DEAdistribution in GA and/or polymeric precursors of GA, and using, having,storing, and/or providing information resulting from such assessment,e.g., for the selection, review (e.g., quality control), and/ormanufacture of GA and/or polymeric precursors of GA. In someembodiments, the present disclosure includes making recommendations forthe selection, review (e.g., quality control), and/or manufacture of GAand/or polymeric precursors of GA based on the methods provided herein.

In some embodiments, methods disclosed herein contemplate observation orassessment of the distribution of one, two, three, or four distinctDEA-modified amino acids (e.g., DEA-modified alanine, DEA-modifiedlysine, DEA-modified glutamic acid, and/or DEA-modified tyrosine),peptides containing DEA-modified alanine, DEA-modified lysine,DEA-modified glutamic acid, and/or DEA-modified tyrosine, and/or totalDEA-modified amino acids and/or peptides in GA and/or polymericprecursors of GA, and use of such observation or assessment to select,monitor, assess, and/or evaluate GA and/or polymeric precursors of GA.For example, in some embodiments, a level of one, two, three, four, ortotal DEA-modified amino acid or peptide in GA or polymeric precursorsof GA can be observed, assessed, and/or compared with a level of one,two, or three other DEA-modified amino acids or peptides in the GA orpolymeric precursors of GA, a level of total DEA-modified amino acid inthe GA or polymeric precursors of GA, and/or a reference value orstandard. In some embodiments, the distribution of one, two, three, orfour distinct DEA-modified amino acids and/or peptides in GA and/orpolymeric precursors of GA can be compared to total DEA in the GA and/orpolymeric precursors of GA. In some embodiments, levels of one, two,three, or four distinct DEA-modified amino acids can be expressed as apercentage of total DEA-modified amino acids in GA and/or polymericprecursors of GA. In some embodiments, action may be taken if thedistribution of DEA-modified amino acids in the GA and/or polymericprecursors of GA comply with or are consistent with a defined orapproved standard. In some embodiments, methods include comparison ofthe distribution of DEA modified amino acids in the GA and/or polymericprecursors of GA with defined or values, standards or levels of DEAmodified amino acids (e.g., Federal Drug Administration (FDA) approvedor commissioned DA approved values, standards or levels). Data pointscan be expressed and/or compared as ratios, levels, relative levels,e.g., as long as the data points can be compared. In some cases,conversion of data points can be performed to facilitate comparison.

As described above, the present disclosure provides methods foraccurately and/or precisely assessing (e.g., measuring, analyzing,detecting, determining, evaluating, estimating, and/or predicting) DEAdistribution e.g., including the level(s) (e.g., level, relative level,concentration, amount, and/or mass) of one or more of DEA-modifiedalanine, DEA-modified lysine, DEA-modified glutamic acid, DEA-modifiedtyrosine, and/or total DEA-modified amino acids in GA and/or a polymericprecursor of GA. Methods can include comparison of such distribution toa reference standard for GA and/or a polymeric precursor of GA (e.g., areference standard showing distribution of one or more of DEA-modifiedalanine, DEA-modified lysine, DEA-modified glutamic acid, andDEA-modified tyrosine in GA and/or a polymeric precursor of GA). BecauseDEA distribution on the C-terminus of a portion of amino acids of GA isa characteristic, hallmark, and/or signature (e.g., a structuralsignature) of GA and/or the GA production process, informationpertaining to the distribution, of DEA can be used to select, monitor,assess, and/or evaluate a GA process and/or batch quality.

In some embodiments, the present disclosure includes assessment oflevel(s) (e.g., level, relative level, concentration, amount, and/ormass) of one or more of DEA-modified alanine, DEA-modified lysine,DEA-modified glutamic acid, and DEA-modified tyrosine, and/or totalDEA-modified amino acids in GA and/or a polymeric precursor of GA to areference value or standard using the ‘isobaric tag for relative andabsolute quantitation (iTRAQ),’ e.g., using mTRAQ®. iTRAQ is based onthe covalent labeling of the N-terminus and side-chain amines ofpeptides from protein digestions with tags of varying mass. Thesesamples can then be pooled and optionally fractionated by nano liquidchromatography and analyzed, for example, by tandem mass spectrometry(MS/MS). The fragmentation of the attached tag generates a low molecularmass reporter ion that can be used to relatively quantify the peptidesand the proteins from which they originated. For example, in someinstances, methods can include labeling GA and/or polymeric precursorsof GA, mixing labeled GA and/or polymeric precursors of GA with areference sample comprising detectably distinct labeling, and analyzingthe mixture. Labeled GA and/or polymeric precursors and reference samplecomprising detectably distinct labeling can be assessed separately ortogether.

Methods discussed herein can be used to identify differences in GAmaterials not observed using conventional methods (e.g., by analysis ofmolar mass and/or amino acid composition). For example, by evaluatingdiethylamide distribution in GA or a polymeric precursor of GA, one canidentify non-conforming compositions during or following the GAmanufacturing process. Alternatively or in addition, methods can be usedto confirm consistency between initiation kinetics in GA productionprocesses (e.g., to determine equivalence or compare consistency ininitiation kinetics between lots of material). For example, comparisonscan be made between two or more batches or lots of GA, e.g.,manufactured by different manufacturers, two or more batches or lots ofGA manufactured at different locations, two or more batches or lots ofGA manufactured at different times, two or more batches or lots of GAmanufactured different processes, and/or two or more batches or lots ofGA manufactured using altered or modified initiation kinetics. Thecompositions and methods herein can also be used in quality control,e.g., to compare and/or confirm batch-to-batch consistency between lotsmade by a consistent process.

As used herein, GA can include GA and/or polymeric precursors of GA,including, but not limited to, compositions comprising GA and/or orpolymeric precursors of GA; a batch or batches, a sample or samples, alot or lots of GA or polymeric precursors of GA, filtrates comprising GAand/or polymeric precursors of GA; and/or mother liquors (e.g., leftafter drying) comprising GA and/or polymeric precursors of GA and/or lowmolecular weight peptides related to GA. A batch of GA and/or apolymeric precursor of GA can be all or part of the product of a GAmanufacturing process (e.g., all or part of a single manufacturingprocess or run). In some cases, one batch is analyzed. In some cases,two or more batches are analyzed. In other cases, multiple samples takenfrom a single batch are analyzed. A composition containing GA can be adrug substance (DS) (also known as an active pharmaceutical ingredient(API), a drug product (DP), or a process intermediate. GA can alsoinclude GA before or after formulation as a drug product.

Polymeric precursors of GA can include but are not limited to, e.g.,Intermediate-1, Intermediate-2, and/or Intermediate-3 described in themanufacturing process above. For example, polymeric precursors of GA caninclude peptides generated during the production of GA, e.g., followingthe initiation step and up to the deprotection step.

The term “amino acid” is not limited to naturally occurring amino acids,but can include amino acid derivatives and/or amino acid analogs. Forexample, in an amino acid copolymer comprising tyrosine amino acids, oneor more of the amino acids can be a homotyrosine. In addition, an aminoacid modified at its C-terminus to incorporate diethylamide is referredto herein as an amino acid or a diethylamide-modified amino acid.Further, a peptide having one or more non-peptide or peptidomimeticbonds between two adjacent residues is included within the scope of thisdisclosure.

As noted above, GA includes four naturally occurring amino acids:L-glutamic acid, L-alanine, L-tyrosine, and L-lysine, each of which canbe modified by the addition of DEA. The present disclosure includesassessing (e.g., measuring, analyzing, detecting, determining,evaluating, estimating, and/or predicting) DEA distribution, e.g., thedistribution (e.g., proportions, levels, relative levels, and/or ratios)of one or more (e.g., including two or more, three or more, and four) ofDEA-modified alanine, DEA-modified lysine, DEA-modified glutamic acid,and DEA-modified tyrosine in GA and/or a polymeric precursor of GA.

In some embodiments, the distribution of DEA in GA or polymericprecursors of GA (e.g., whether determined, provided, or obtained) canbe compared to the values provided in Table 1. In some instances, thedistribution of DEA in GA or a polymeric precursor of GA can correlatewith, can be equal (e.g., about equal), and/or can be equivalent (e.g.,about equivalent) to a level or ratio shown in Table 1.

Values in Table 1 relate to DEA distribution in GA and/or polymericprecursors of GA, including DEA-modified amino acids, DEA-modifiedpeptides, and/or combinations of DEA-modified amino acids andDEA-modified peptides. These values are expressed as percent orpercentages (%), which, as a skilled practitioner will appreciate,indicate a level relative to total (e.g., the actual (e.g., measured),predicted, or theoretical total) DEA-modified amino acid in the sample(including DEA-modified amino acids and/or DEA-modified peptides). Allvalues shown in Table 1 are approximate or about and can include, e.g.,+/−10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, 0.2, 0.15, 0.1,0.075, 0.05, 0.025, 0.01.

Table 1 includes value (%) ranges and range mean (shown in parentheses)for or relating to each of Tyr-DEA, Glu-DEA, Lys-DEA, and Ala-DEA, shownas W (superscript), Y (superscript), and Z (superscript). As used inTable 1, W refers to mean (< >) minimum and mean maximum values, derivedfrom Table 2 (see Example 1). Y refers to the minimum W value multipliedby 0.75 and the maximum W value multiplied by 1.25. Z refers to theminimum W value multiplied by 0.9 and the maximum W value multiplied by1.1.

Shaded rows in Table 1 show ratios calculated from W values. a(superscript) ratios are normalized against the mean of the w range forTyr-DEA. b (superscript) ratios are normalized against the mean of the wrange for Glu-DEA. c (superscript) ratios are normalized against themean of the w range for Ala-DEA. “X” indicates data points that can becompared.

Use of Table 1 can include, but is not limited to, for example,comparison of a value or range shown in rows L with DEA distribution inGA and/or polymeric precursors of GA; and/or comparison of one or moreof the values or ranges shown in rows L and one or more of the values orranges shown in columns P with DEA distribution in GA and/or a polymericprecursor of GA. In some instances, W values in column L are comparedwith W values in rows P, Y values in column L are compared with Y valuesin rows P, and/or, Z values in column L are compared with Z values inrows P. In some instances, a value or range representing any one or more(e.g., one, two, three, four) of Tyr-DEA, Glu-DEA, Lys-DEA, and/orAla-DEA can be compared to any one or more other feature of GA,including, for example, molecular weight, chain ends, total number ofchains, and/or total DEA, etc.

For example, use of Table 1 can include comparison of data points forone, two, three, four, and/or total DEA-modified amino acids shown inTable 1 with DEA distribution in GA and/or polymeric precursors of GA.By way of non-limiting example, use of Table 1 can include comparison ofa data point for one DEA-modified amino acid shown in Table 1, e.g.,expressed as a percentage of total DEA, with a level of the sameDEA-modified amino acid, e.g., expressed in the same or a comparableformat, in GA and/or polymeric precursors of GA.

For instance, as conveyed by Table 1, comparison can include comparisonof 60-76% Ala-DEA (see column L, Ala-DEA, W range) with DEA distributionin GA and/or a polymeric precursor thereof. Alternatively or inaddition, comparison can include comparison of 60-76% Ala-DEA (seecolumn L, Ala-DEA, W range) and 11-17% Lys-DEA (see rows P, Lys-DEA, Wrange) with DEA distribution in GA and/or a polymeric precursor thereof.In some instances, correlation, equality, and/or equivalence betweensuch values in Table 1 and DEA distribution in the GA and/or thepolymeric precursor thereof can facilitate selection of the GA and/orthe polymeric precursor thereof. As indicated above, data points shownin Table 1 can be compared to levels of DEA-modified amino acids in GAand/or polymeric precursors of GA that have been determined (e.g.,physically determined), predicted, estimated (e.g., based on averagelevels of DEA-modified amino acids in two or more lots or batches of GAand/or polymeric precursors of DEA; by using diethylamine recovered fromGA manufacture to calculate or estimate total DEA incorporation into theGA and/or polymeric precursors of GA; and/or by using diethylaminerecovered from GA manufacture to calculate or estimate DEA-modifiedamino acids in GA and/or polymeric precursors of GA), provided, and/orrecorded.

Thus, as discussed in more detail below, Table 1 can be used to assessDEA distribution in GA and/or a polymeric precursor thereof by comparingthe DEA distribution in the GA and/or a polymeric precursor thereof withthe values in Table 1.

TABLE 1 P Ala-DEA Glu-DEA 59.5-76.1 Lys-DEA 9.9-15.0 Tyr-DEA (67.8)11.3-17.3 (14.3) (12.5) 4.8-7.2 (6.0) 60-76 (68)^(w) 11-17 (14)^(w)10-15 (13)^(w) 5-7 (6)^(w) 45-95 (70)^(y) 9-22 (15)^(y) 7-19 (13)^(y)4-9(6)^(y) Total % 54-84 (69)^(z) and/or 10-19 (15)^(z) and/or 9-17(13)^(z) and/or 4-8(6)^(z) and/or 100 L Tyr- 4.8-7.2 (6.0) X X X — X(Optionally DEA 5-7 (6)^(w) compare 4-9 (6)^(y) with P) 4-8 (6)^(z)1^(a) 10  2 2 — Glu- 9.9-15.0 (12.5) X X — X X DEA 10-15 (13)^(w) 7-19(13)^(y) 9-17 (13)^(z) 1^(b) 4 0.9 — 0.5 Lys- 11.3-17.3 (14.3) X — X X XDEA 11-17 (14)^(w) 9-22 (15)^(y) 10-19 (15)^(z) 1^(c) 5 — 1 0.5 Ala-59.5-76.1 (67.8) — X X X X DEA 60-76 (68)^(w) 45-95 (70)^(y) 54-84(69)^(z) 1^(d) — 0.2 0.2 0.1In some instances, the values shown in Table 1 are reference values(e.g., a specification for commercial release of GA) and methods caninclude comparing the distribution of DEA in GA and/or a polymericprecursor to the reference values in Table 1. For example, the methodsherein generally include obtaining or providing GA and/or a polymericprecursor and/or obtaining data regarding DEA distribution in GA and/ora polymeric precursor of GA, assessing the distribution of DEA therein,and comparing the distribution of DEA to Table 1. Distribution of one ormore, including all, of Ala-DEA, Lys-DEA, Glu-DEA, Tyr-DEA, and/or totalDEA can be assessed. Total DEA is the sum of DEA-modified amino acids inGA and/or polymeric precursors of GA. Total DEA can be actual (e.g.,based on one or more measurements of DEA in the GA and/or polymericprecursor) or theoretical (e.g., based on an average value for total DEAin GA and/or a specific stage in the GA manufacturing process (e.g.,total DEA in Intermediate-1, Intermeidate-2, and/or Intermediate-3)).DEA-modified amino acids can be measured separately and/or together andthe sum of DEA-modified amino acids can be determined therefrom. Priorto measuring total DEA, DEA can be removed from amino acids and/orpeptides, e.g., by hydrolysis. For example, DEA can be measured afterhydrolysis of GA and/or polymeric precursors of GA. For polymericprecursors of GA, total DEA can include the sum of DEA-modified aminoacids at a defined stage in the GA manufacturing process.

DEA distribution can be assessed by review of DEA levels in GA orpolymeric precursors. Levels can include the level of one or more ofAla-DEA, Lys-DEA, Glu-DEA, Tyr-DEA, and/or total DEA as independentvalues (e.g., mass or volume) or as relative values (e.g., the level ofone or more DEA modified amino acid relative to the level of one or moreof the other DEA modified amino acids or total DEA (e.g., percentage orconcentration)). Levels can include: the level of Ala-DEA or the levelof Ala-DEA and the level of one or more of Lys-DEA, Glu-DEA, Tyr-DEA,and/or total DEA; the level of Lys-DEA or the level of Lys-DEA and thelevel of one or more of Ala-DEA, Glu-DEA, Tyr-DEA, and/or total DEA; thelevel of Glu-DEA, or the level of Glu-DEA and the level of one or moreof Lys-DEA, Ala-DEA, Tyr-DEA, and/or total DEA; and/or the level ofTyr-DEA, or the level of Tyr-DEA and the level of one or more ofLys-DEA, Ala-DEA, Ala-DEA, and/or total DEA.

Levels of DEA in the GA and/or a polymeric precursor can be expressed inany suitable units. For example, levels can be expressed as percentvalues (as exemplified in Table 1) and/or as ratios of two or more,three or more, four or more, or all of Ala-DEA, Lys-DEA, Glu-DEA,Tyr-DEA, and/or total DEA. In addition, units can be converted tofacilitate comparison with Table 1 using techniques known in the artand/or reasonable skill. Values can also be represented by any otheruseful parameter by converting the distribution, proportions, relativelevels, and/or ratios of diethylamide into such parameters. For example,the values can be converted to relative molar amounts and/or mole % orpercent of chains. Unit conversion is not required if equivalence can bedetermined.

GA and/or a polymeric precursor of GA can be selected if thedistribution of DEA in the GA or the polymeric precursor of GAcorrelates with, is equal (e.g., about equal) to, and/or is equivalent(e.g., about equivalent) to, a level or ratio shown in Table 1. Forexample, methods can include comparing the distribution of DEA in GAand/or a polymeric precursor of GA with Table 1, and selecting the GAand/or the polymeric precursor if the distribution of DEA in the GA orthe polymeric precursor of GA correlates with, is equal (e.g., aboutequal) to, and/or is equivalent (e.g., about equivalent) to, a level orratio shown in Table 1.

As shown in Table 1, the distribution, of one or more DEA-modified aminoacids in GA and/or its polymeric precursors can be, for example:

a distribution of DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about62-68%:12-15%:14-16%:6-7%, e.g., wherein the sum of the distribution is100%;

a distribution of DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about47-85%:9-19%:11-20%:5-9%, e.g., wherein the sum of the distribution is100%;

a distribution of DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about56-75%:11-17%:13-18%:5-8%, e.g., wherein the sum of the distribution is100%;

a ratio of two or more of DEA-modified alanine:DEA-modifiedlysine:DEA-modified glutamic acid:DEA-modified tyrosine, wherein thevalue for DEA-modified alanine is about 10, the value for DEA-modifiedlysine is about 2, the value for DEA-modified glutamic acid is about 2,and the value of DEA-modified tyrosine is about 1, wherein about can be±0.1, ±0.2, ±0.3, ±0.4, and ±0.5; a ratio of about DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid:DEA-modifiedtyrosine of or of about (e.g., wherein about can be ±0.1, ±0.2, ±0.3,±0.4, and ±0.5) 10:2:2:1;

a ratio of two or more of DEA-modified alanine:DEA-modifiedlysine:DEA-modified glutamic acid:DEA-modified tyrosine, wherein thevalue for DEA-modified alanine is about 4, the value for DEA-modifiedlysine is about 0.9, the value for DEA-modified glutamic acid is about1, and the value of DEA-modified tyrosine is about 0.5, wherein aboutcan be ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5;

a ratio of about DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about (e.g., wherein aboutcan be ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5) 4:0.9:1:0.5;

a ratio of two or more of DEA-modified alanine:DEA-modifiedlysine:DEA-modified glutamic acid:DEA-modified tyrosine, wherein thevalue for DEA-modified alanine is about 5, the value for DEA-modifiedlysine is about 1, the value for DEA-modified glutamic acid is about 1,and the value of DEA-modified tyrosine is about 0.5, wherein about canbe ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5;

a ratio of about DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about (e.g., wherein aboutcan be ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5) 5:1:1:0.5;

a ratio of two or more of DEA-modified alanine:DEA-modifiedlysine:DEA-modified glutamic acid:DEA-modified tyrosine, wherein thevalue for DEA-modified alanine is about 1, the value for DEA-modifiedlysine is about 0.2, the value for DEA-modified glutamic acid is about0.2, and the value of DEA-modified tyrosine is about 0.1, wherein aboutcan be ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5; and/or

a ratio of about DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine of or of about (e.g., wherein aboutcan be ±0.1, ±0.2, ±0.3, ±0.4, and ±0.5) 1:0.2:0.2:0.1.

In some embodiments, methods do not include assessment of diethylamineand/or distribution of DEA-modified amino acids in GA and/or a polymericprecursor of GA does not include diethylamine. Unless methods includecleaving DEA from the C-terminal of DEA-modified amino acids, methods donot include assessing DEA not associated with amino acid (free DEA).

Selection of GA and/or polymeric precursors of GA can include selecting(e.g., for use or further processing) a sample of GA or a polymericprecursor of GA based on distribution of DEA-modified amino acids in thesample (e.g., based on comparison of distribution of DEA-modified aminoacids in the sample with at least one (e.g., one, two, three, four, ormore) reference value(s) shown in Table 1). For example, the methods canbe used to: select a sample of GA or a polymeric precursor thereof forfurther use; select (e.g., as suitable for sale or for administration(e.g., injection) to a human) a sample of GA or a polymeric precursorthereof; classify, accept, release, process into drug product a sampleof GA or a polymeric precursor thereof; select a sample of GA or apolymeric precursor thereof for shipment, moving to a new location,formulating, labeling, packaging, selling, offering for sale, releasinginto commerce; and/or select a sample of GA or a polymeric precursorthereof for use in a manufacturing process for GA. Methods can alsoinclude assessment of Intermediate-1 and use of the Intermediate-1 in amanufacturing process for GA precursor if the level of one or more ofAla-DEA, Lys-DEA, Glu-DEA, and/or Tyr-DEA in the Intermediate-1 is equal(e.g., about equal) to, equivalent (e.g., about equivalent) to, and/orconsistent with a reference value, wherein the reference value is areference value disclosed in Table 1; assessment of Intermediate-2 anduse of the Intermediate-2 in a manufacturing process for GA precursor ifthe level of one or more of Ala-DEA, Lys-DEA, Glu-DEA, and/or Tyr-DEA inthe Intermediate-2 is equal (e.g., about equal) to, equivalent (e.g.,about equivalent) to, and/or consistent with a reference value, whereinthe reference value is a reference value disclosed in Table 1; and/orassessment of Intermediate-3 and use of the Intermediate-3 in amanufacturing process for GA precursor if the level of one or more ofAla-DEA, Lys-DEA, Glu-DEA, and/or Tyr-DEA in the Intermediate-3 is equal(e.g., about equal) to, equivalent (e.g., about equivalent) to, and/orconsistent with a reference value, wherein the reference value is areference value disclosed in Table 1.

In some embodiments, a sample of GA or a polymeric precursor of GA canbe selected if the distribution of DEA in the GA has a preselectedrelationship with, is equal (e.g., about equal) to, is equivalent (e.g.,about equivalent) to, and/or is consistent with the levels or ratiosshown in Table 1. In some embodiments, methods can include selecting GAor a polymeric precursor if the level of one or more of Ala-DEA,Lys-DEA, Glu-DEA, and/or Tyr-DEA is equal (e.g., about equal) to,equivalent (e.g., about equivalent) to, and/or consistent with areference value, wherein the reference value is a reference valuedisclosed in Table 1.

The methods described herein can also include selecting to discard,withhold, reprocess through a previous manufacturing step, ordiscontinue use of, GA or a polymeric precursor of GA, for example, ifthe distribution of DEA in the GA or the polymeric precursor do not meeta preselected relationship, are not equal to, are not equivalent with,and/or are not consistent with the levels shown in Table 1.

Methods for Determining DEA Distribution

In some embodiments, the present disclosure includes measuring,assessing, determining or detecting one or more of DEA-modified alanine,DEA-modified lysine, DEA-modified glutamic acid, DEA-modified tyrosine,and/or total DEA-modified amino acid in a sample of GA and/or apolymeric precursor of GA. Accordingly, the disclosure provides methodsfor accurately and/or precisely measuring, assessing, determining ordetecting one or more of DEA-modified alanine, DEA-modified lysine,DEA-modified glutamic acid, DEA-modified tyrosine, and/or totalDEA-modified amino acid in a sample of GA and/or a polymeric precursorof GA. Other methods not explicitly disclosed herein may also be used solong as they allow at least detection (e.g., specific detection) of oneor more of DEA-modified alanine, DEA-modified lysine, DEA-modifiedglutamic acid, DEA-modified tyrosine, and/or total DEA-modified aminoacid in a sample of GA and/or a polymeric precursor of GA.

Referring to FIG. 1, a flow diagram is provided illustrating onenon-limiting exemplary embodiment of a method for assessing DEAdistribution. In STEP O, a sample comprising GA or a polymeric precursorthereof is provided or obtained. In STEP 1, the cleaving step, the GA ora polymeric precursor thereof in the sample is cleaved to produce in thesample shorter or smaller fragments (e.g., smaller peptide fragments) ofthe GA or the polymeric precursor without removing (e.g., withoutsubstantially removing or with consistently minimal removal of)C-terminal DEA from the GA or the polymeric precursor. Fragments caninclude, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acidresidues. In STEP 2, the labeling step, detectable standards (e.g.,DEA-modified detectable peptides that represent some or all of thefragments produced in STEP 1) are added (e.g., in known amounts) to thesample containing shorter or smaller fragments of GA or the precursor toproduce a labeled sample. In STEP 3, the separation step, fragments andlabeled standards in the labeled sample are separated intosubpopulations with shared or common properties. In STEP 4, thedetection step, the subpopulations are assessed using method capable ofdetecting and distinguishing the detectable standards and the fragmentsof GA and the polymeric precursor. The detectable standards can then besubtracted and the distribution of diethylamide-modified amino acidsdetermined.

In some embodiments, methods can include one or more (e.g., one, two,three, all, or substantially all) of STEP O, STEP 1, STEP 2, STEP 3, andSTEP 4, e.g., performed in any order that results in determination ofDEA distribution, including variations and/or modifications of STEPS 1,2, 3, and/or 4.

Step O

In some embodiments, methods include providing or obtaining a sample(s)comprising GA or a polymeric precursor thereof can include GA and/orpolymeric precursors of GA, e.g., as described above, including, but notlimited to, compositions comprising GA and/or or polymeric precursors ofGA; a batch or batches, a sample or samples, and/or a lot or lots of GAor a polymeric precursors of GA, filtrates comprising GA and/orpolymeric precursors of GA; and/or mother liquors (e.g., left afterdrying) comprising GA and/or polymeric precursors of GA. Samples may bechemically modified to convert diethylamides into new chemical groups.Such methods can include, but are not limited to, modification ofdiethylamides into chemically distinct groups resistant to cleavage(e.g., reduction of diethylamides to alcohols).

Step 1

In some embodiments, methods include STEP 1, the cleaving step. In someembodiments, STEP 1 can be performed using any treatment or method, orcombination thereof that can cleave (e.g., specifically cleave) peptidebonds without removing diethylamide (e.g., without substantiallyremoving diethylamide). Such treatments or methods can yield singleamino acid residues and/or cleaved peptides, e.g., peptides containingfewer amino acid residues than would be present in the same peptideabsent the treatment of method. Such cleaved peptides can include 2, 3,4, 5, 6, 7, 8, 9, 10, or more amino acid residues.

Each of the species resulting from the cleaving step (e.g., the speciesof single amino acid residues and/or the species of cleaved peptides),which can be collectively referred to as fragments, can includesub-species, e.g., DEA-modified fragments, that include DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid: DEA-modifiedtyrosine and cleaved peptides that include DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid:DEA-modifiedtyrosine. For example, cleaved peptides can include 2, 3, 4, 5, 6, 7, 8,9, 10, or more amino acid residues, wherein the C-terminal amino acidresidue is DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine. Suitable methods and treatments caninclude, e.g., one or more of enzymatically, chemically, and/orphysically cleaving the GA. In some embodiments, the average number ofamino acids these DEA-modified fragments is 4 or less amino acids, 3 orless amino acids, or two or less amino acids.

Exemplary enzymatic methods for use in STEP 1 can include contacting theGA or the polymeric precursor with an enzyme that can cleave or digestpeptide bonds in the GA or the polymeric precursor without removing orsubstantially removing DEA therefrom. Suitable enzymes can include oneor more proteases, and suitable proteases can include, but are notlimited to, for example, one or more of trypsin, chymotrypsin, elastase,ficin, papain, pepsin, plasmin, thermolysin, endopeptidase, proteinaseK, ox bile, lemon pectin, horseradish peroxidase, glu-c, endo lys-C,carboxypeptidase, calpain, and subtilisin. In some embodiments, theenzyme is removed following cleavage of the GA, e.g., by filtration.

Exemplary chemical methods for use in STEP 1 can include contacting ortreating the GA or the polymeric precursor with a chemical or compoundthat can cleave peptide bonds in the GA or the polymeric precursorwithout removing or substantially removing DEA therefrom. Suitablechemicals can include, but are not limited to, e.g., one or more of astrong acid, an alkali base, and phenylisothiocyanate.

Exemplary physical methods for use in STEP 1 can include treating the GAor the polymeric precursor using conditions that cleave or digestpeptide bonds in the GA or the polymeric precursor without removing orsubstantially removing DEA therefrom. Suitable conditions or treatmentscan include, but are not limited to, e.g., one or more of boiling,sonicating, and shearing.

In some embodiments, STEP 1 can include producing a sample that containsonly DEA-modified amino acids and/or DEA-modified peptides. For example,the methods can include isolating or purifying DEA-modified amino acidsand/or DEA-modified peptides; removing amino acids and/or peptides thatdo not include a DEA-modified amino acid; and/or removing diethylamine.

Step 2

In some embodiments, methods can include STEP 2, a labeling step. Insome embodiments, STEP 2 can include supplementing or adding detectablestandards to the sample. The detectable standards can include moleculesthat are representative of each of the DEA-modified amino acid residuesand/or DEA-modified cleaved peptides produced or expected to be producedby the cleaving step. In some instances, such molecules can includeDEA-modified amino acids or DEA-modified peptides that are identical(e.g., chemically identical) to the fragments obtained in the cleavingstep, but that can be distinguished from those fragments (e.g., due toone or more distinct or unique properties (e.g., the presence of one ormore detectable markers)) and/or that can be detected. In someinstances, the detectable standards can be chemically distinct (e.g.,can include chemically distinct DEA, chemically distinct carboxylates,and/or chemically distinct peptide bonds).

In some embodiments, the detectable standards can have substantially thesame retention time under chromatography (e.g., liquid chromatography)as the fragments produced in the cleavage step, but with distinct ordistinguishable peaks on a chromatograph. For example, the detectablestandards can be isotopically labeled DEA-modified amino acids and/orDEA-modified peptides that represent one or more of the DEA-modifiedamino acid residues and/or DEA-modified cleaved peptides produced orexpected to be produced by the cleaving step. In some instances,isotopically labeled DEA-modified amino acids and/or DEA-modifiedpeptides that represent each of the DEA-modified amino acid residuesand/or DEA-modified cleaved peptides produced or expected to be producedby the cleaving step. In some cases, isotopically labeled DEA-modifiedamino acids or peptides can include, e.g., 1, 2, 3, or more amino acids,wherein one of the amino acids (e.g., the C-terminal amino acid) is aDEA-modified amino acid. In some cases, the concentration or amount oflabeled standard added or in the sample is known, e.g., to facilitatelater accounting for or subtraction of the labeled standards.

Alternatively or in addition, STEP 2 can include manipulation ofDEA-modified amino acids to produce DEA-modified amino acids that can bedistinguished from other DEA-modified amino acids and/or that can bedistinguished from amino acids and/or peptides that do not include DEA.For example, DEA-modified alanine can be modified to be distinguishablefrom one or more of DEA-modified lysine, DEA-modified glutamic acid,and/or DEA-modified tyrosine; DEA-modified lysine can be modified to bedistinguishable from one or more of DEA-modified alanine, DEA-modifiedglutamic acid, and/or DEA-modified tyrosine; DEA-modified glutamic acidcan be modified to be distinguishable from one or more of DEA-modifiedalanine, DEA-modified lysine, and/or DEA-modified tyrosine; DEA-modifiedtyrosine can be modified to be distinguishable from one or more ofDEA-modified alanine, DEA-modified glutamic acid, and/or DEA-modifiedlysine; and/or one or more of DEA-modified alanine, DEA-modified lysine,DEA-modified glutamic acid, and/or DEA-modified tyrosine can be modifiedto be distinguishable from amino acids and/or peptides that do notinclude DEA. Such methods can include, but are not limited to, forexample, modifying DEA to chemically distinct groups, changingcarboxylates to chemically distinct groups, and/or changing peptidebonds to chemically distinct groups.

Step 3

In some embodiments, methods can include STEP 3, a separation step. Insome embodiments, STEP 3 can include any method whereby fragments of GAare separated into subpopulations of macromolecules. For example, theseparation can be based on one or more of a chemical, physical, and/orfunctional property shared by a class of macromolecules within thefragments of GA, e.g., size, charge, hydrophobicity, or any other sharedproperty. In some instances, the fragments can be separated intosubpopulations of amino acids and peptides that includediethylamide-modified C-termini and those that do not. The subpopulationof fragments that include diethylamide-modified C-termini can also beseparated (e.g., further separated) into DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid:DEA-modifiedtyrosine. Suitable methods can include, but are not limited to, e.g.,analysis of migration through a gel, size, molecular weight, migrationthrough an applied electrical field, charge, hydrophobicity, boilingpoint, and solubility, as well as chromatographically separating by sizeand/or charge, and obtaining the fragments of GA and the labeledstandards. Suitable methods for performing this step can include, butare not limited to, for example, gas chromatography (GC), GC-MS, liquidchromatography, liquid chromatography mass spectroscopy, ionchromatography, mass spectroscopy, nuclear magnetic resonance (NMR),antibody methods, Raman spectroscopy, capillary electrophoresis,multidimensional NMR spectroscopy, extraction, and/or precipitation.Single or multiple separation steps can be used.

Step 4

In some embodiments, methods can include STEP 4, a detection step. Insome embodiments, STEP 4 can include any method that can detect thefragments of GA and the labeled standards and that can distinguish thefragments of GA from the labeled standards. Suitable methods caninclude, but are not limited to, for example, mass spectroscopyanalysis, MRM detection, tandem mass spectrophotometry (MS/MS), NMRanalysis, infrared spectroscopy, gel electrophoresis, emissionspectroscopy, UV-vis spectroscopy, Raman spectroscopy, fluorescencespectroscopy, emission spectroscopy, and/or antibody detection. In someinstances, the detection step includes MS-MRM detection, e.g., whichselects targeted parent ions for fragmentation and then searches forspecific fragment (daughter) ions. Only species that match the parentand/or daughter ions of the targeted species are detected, giving riseto high specificity with low background.

The detection step can provide levels of DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid:DEA-modifiedtyrosine in the fragments of GA and in the GA. Such levels can includelevels of the labeled standard. However, as the labeled standard can bedistinguished from unlabeled DEA-modified amino acids and because theconcentration or amount of labeled standard added is known, labeledstandard can be subtracted. In embodiments where isotopically labeledDEA-modified amino acids or peptides are used as the labeled standards,the standards can be designed to elute at the same time and/or ionizewith the same efficiency as the DEA modified amino acids in thefragments of GA. However, because of the isotopic enrichment, thestandards have distinct MRM signatures from the target species. Thus, insuch embodiments, the relative difference in area between the MRMsignals of the standards to the MRM signals of the target species aredirectly proportional to the differences in concentrations. Therefore,since the concentrations of the standards are known, the concentrationsof the target species can be directly determined from the ratios of theMRM signals.

Detected levels of DEA-modified alanine:DEA-modified lysine:DEA-modifiedglutamic acid:DEA-modified tyrosine can include various species. Suchspecies can include, for example, DEA-modified amino acids andDEA-modified peptides. DEA modified peptides can include 2 or more aminoacids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids).DEA-modified peptides can be referred to collectively by reference totheir DEA-modified amino acid, as is done throughout the presentdisclosure. For example, any peptides with a C-terminal DEA modifiedalanine can be referred to as a diethylamide-modified alanine peptide orAla-DEA. Alternatively or in addition, the sum of each of the distinctthe levels of each of the diethylamide modified peptides can bedetermined to calculate total DEA-modified alanine:DEA-modifiedlysine:DEA-modified glutamic acid:DEA-modified tyrosine in the GA orpolymeric precursor.

Alternatively or in addition, methods can include direct detection ofDEA, e.g., with or without use of the cleaving and/or labeling stepsdiscussed above. Such methods include detection of DEA associated withamino acid (e.g., DEA-modified amino acids and/or peptides (bound DEA))and DEA not associated with amino acid (free DEA). Such methods caninclude a first optional separation to obtain a population ofDEA-modified peptides from GA or a polymeric precursor of GA, and asecond separation to obtain distinct populations of peptides containingC-terminal Ala-DEA, Lys-DEA, Glu-DEA or Tyr-DEA. In some instances, anyfree DEA can be removed before detection. DEA can then be separatelydetected in one or more, including all, of the distinct populations,e.g., without the need to further determine the species of DEA-modifiedpeptide (e.g., DEA detected in the Ala-DEA population is DEA associatedwith Ala). Detection can include direct assessment of bound DEA usingtechniques known in the art and disclosed herein. Detection can alsoinclude removal (e.g., cleavage) of bound DEA from DEA-modified peptidesusing techniques known in the art and disclosed herein to obtain freeDEA. Free DEA can then be detected using methods known in the art.Detected DEA from one or more of the populations can then be compared toa reference value. GA or polymeric precursors can be selected if thedistribution of DEA is equal (e.g., about equal) to, equivalent (e.g.,about equivalent) to, and/or consistent with a reference value, whereinthe reference value is a reference value disclosed in Table 1.

In some embodiments, DEA detected using the methods disclosed herein canbe compared to diethylamine. Methods for detection of diethylamine areknown in the art (see, e.g., US 2007/0054857). The steps recited hereindo not indicate or imply order. Accordingly, the steps can be performedcontemporaneously or in any order that allows a result to be obtained.For example, the cleaving step can be performed before, after, or aboutsimultaneously (e.g., simultaneously) with the labeling step. Similarly,the separating step can be performed before or about simultaneously(e.g., simultaneously) with the detecting step. These values can berepresented as distributions, proportions, relative levels, and/orratios of DEA in GA or polymeric precursor (see above).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Comparison of Reference Listed Drug

As shown herein, the distribution of diethylamide (DEA) in the C-terminiof glatiramer acetate (GA) and the sequence context of DEA in GA, namelythe identity of the adjacent amino acid residue, is a direct consequenceof the initiation kinetics applied in the manufacturing of GA. Thedifferential reactivity of the NCAs (initiation rate constants) andproportions of NCAs control the proportions of amino acid diethylamidesproduced. The initiation step is the only step in the manufacturingprocess that creates diethylamides. Subsequent processing can removesome of the diethylamides.

Multiple lots of COPAXONE™ were analyzed for DEA-modifiedalanine:DEA-modified lysine:DEA-modified glutamic acid:DEA-modifiedtyrosine using the method illustrated in FIG. 1. The mean, minimum, andmaximum values obtained from this analysis are shown in Table 2.

TABLE 2 Comparison of Multiple Lots of Glatiramer Acetate RLD % Ala-DEA% Lys-DEA % Glu-DEA % Tyr-DEA Mean 67.8 14.3 12.5 6.0 Minimum 59.5 11.39.9 4.8 Maximum 76.1 17.3 15.0 7.2

This data suggests that the distribution of DEA within GA can serve as asignature of GA. The consistency of these data also suggests that themethods disclosed herein are accurate.

It should be noted that the ratio of DEA modified amino acids differsfrom the ratio of amino acids in GA as shown in Table 3. For example,while Lys represents 33.8% of the amino acids in GA, Lys-DEA representsonly 11.3-17.3% of the DEA modified amino acids in GA. In contrast, Alarepresents 42.7% of the amino acids in GA, but Ala-DEA represents59.5-76.1% of the DEA modified amino acids in GA.

TABLE 3 Comparison of amino acid ratios and DEA-modified amino acidsAmino acid % in GA % DEA modified in GA (mean) Glu 14.1  9.9-15.0 (12.5)Ala 42.7 59.5-76.1 (67.8) Tyr 9.5  4.8-7.2 (6.0) Lys 33.8 11.3-17.3(14.3)

Methods disclosed herein were also shown to be precise and sensitive.For example, the process was shown to be robust in that changes indigestion and analytical conditions could be accommodated withoutadversely impacting sensitivity, precision, and/or accuracy. Use ofdifferent lots of columns and/or enzyme also did not adversely impactsensitivity, precision, and/or accuracy. This suggests that the methodsdisclosed herein are precise. Furthermore, changing the initial NCAcharge proportions produced significant changes in the diethylamideproportions in the expected directions (e.g. decreasing the initialcharge of Ala NCA decreased the proportion of Ala-DEA in the product).This suggests the methods are capable of detecting changes in initiationkinetics used in the manufacture of GA.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method of manufacturing glatiramer acetate drugproduct, the method comprising: preparing an amino acid copolymer ofL-glutamic acid, L-alanine, L-Lysine, and L-tyrosine; measuring, in atleast one sample of the copolymer, the levels of at least two, three, orfour individual diethylamide-modified amino acids selected from thegroup consisting of: diethylamide-modified alanine,diethylamide-modified lysine, diethylamide-modified glutamic acid, anddiethylamide-modified tyrosine; and processing the copolymer to produceglatiramer acetate drug product if the measured levels of at least oneof the at least two, three, or four measured individualdiethylamide-modified amino acids meets commercial release specification(i), (ii), (iii), or (iv): (i) the level of diethylamide-modifiedalanine in the sample is 59.5-76.1% of the total diethylamide-modifiedamino acids in the sample on a mol percent basis; (ii) the level ofdiethylamide-modified lysine detected in the sample is 11.3-17.3% of thetotal diethylamide-modified amino acids in the sample on a mol percentbasis; (iii) the level of diethylamide-modified glutamic acid detectedin the sample is 9.9-15.0% of the total diethylamide-modified aminoacids in the sample on a mol percent basis; or (iv) the level ofdiethylamide-modified tyrosine detected in the sample is 4.8-7.2% of thetotal diethylamide-modified amino acids in the sample on a mol percentbasis, thereby manufacturing glatiramer acetate drug product.
 3. Themethod of claim 2 wherein: the step of preparing comprisesco-polymerizing N-carboxy anhydrides of L-alanine, benzyl-protectedL-glutamic acid, trifluoroacetic acid (TFA)-protected L-lysine, andL-tyrosine to generate a first material; treating the first material todeprotect the benzyl-protected L-glutamic acid therein and partiallydepolymerize the first material, thereby generating a second material;and purifying and optionally drying the second material; or purifyingand optionally drying the second material to produce a purified andoptionally dried second material and treating the purified andoptionally dried second material with aqueous piperidine to deprotectthe TFA-protected L-lysine, to thereby produce the copolymer ofL-glutamic acid, L-alanine, L-lysine, and L-tyrosine.
 4. The method ofclaim 2 wherein: the step of preparing comprises co-polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a first material; treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and partially depolymerize thefirst material, thereby generating a second material; purifying anddrying the second material to produce a purified and dried secondmaterial; treating the purified and dried second material with aqueouspiperidine to deprotect the TFA-protected L-lysine to produce a thirdmaterial; and purifying and optionally drying the third material, tothereby produce the copolymer of L-glutamic acid, L-alanine, L-lysine,and L-tyrosine.
 5. The method of claim 4, comprising purifying anddrying the third material, to thereby produce the copolymer ofL-glutamic acid, L-alanine, L-lysine, and L-tyrosine.
 6. The method of4, wherein purifying the third material comprisesdiafiltration/ultrafiltration.
 7. The method of claim 2, comprisingprocessing the copolymer to produce glatiramer acetate drug product ifthe measured level of two of the at least two, three, or four measuredindividual diethylamide-modified amino acids meets commercial releasespecification (i), (ii), (iii), or (iv).
 8. The method of claim 2,comprising processing the copolymer to produce glatiramer acetate drugproduct if the measured level of three of the at least two, three, orfour measured individual diethylamide-modified amino acids meetscommercial release specification (i), (ii), (iii), or (iv).
 9. Themethod of claim 2, comprising processing the copolymer to produceglatiramer acetate drug product if the measured level of four of the atleast two, three, or four measured individual diethylamide-modifiedamino acids meets commercial release specification (i), (ii), (iii), or(iv).
 10. The method of claim 3, wherein co-polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a first material comprises contacting the N-carboxy anhydridesof L-alanine, benzyl-protected L-glutamic acid, trifluoroacetic acid(TFA)-protected L-lysine, and L-tyrosine with diethylamine.
 11. Themethod of claim 3, wherein treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and to partially depolymerizethe first material comprises the first material with phenol treatedHBr/acetic acid.
 12. The method of claim 4, wherein co-polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a first material comprises contacting the N-carboxy anhydridesof L-alanine, benzyl-protected L-glutamic acid, trifluoroacetic acid(TFA)-protected L-lysine, and L-tyrosine with diethylamine.
 13. Themethod of claim 4, wherein treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and to partially depolymerizethe first material comprises the first material with phenol treatedHBr/acetic acid.
 14. The method of claim 2, wherein the glatirameracetate drug product has an Mp of 5000-9000 Da.
 15. The method of claim2, wherein the measuring step comprises: (i) cleaving the glatirameracetate to generate peptide fragments; (ii) combining the peptidefragments with detectably labeled DEA-modified peptide standards; (iii)fractioning the mixture of peptide fragments and detectably labeledDEA-modified peptide standards to generate subpopulations; and (iv)detecting the peptide fragments and detectably labeled DEA-modifiedpeptide standards in one or more subpopulations by a method selectedfrom mass spectroscopy analysis, MRM detection, tandem massspectrophotometry (MS/MS), and NMR.
 16. The method of claim 15, whereinthe peptide fragments comprise peptides and amino acids.
 17. The methodof claim 16, wherein the average length of the peptide fragments is lessthan 4 amino acids.
 18. The method of claim 15, wherein detectablylabeled DEA-modified peptide standards comprise DEA-modified peptidesand DEA-modified amino acids.
 19. The method of claim 2, furthercomprising packaging, labeling, or releasing into commerce theglatiramer acetate drug product.
 20. The method of claim 2, furthercomprising offering for sale or selling the glatiramer acetate drugproduct.
 21. The method of claim 19, further comprising offering forsale or selling the glatiramer acetate drug product.